Antigenic variation plays a vital role in the pathogenesis of many infectious agents, most notably bacteria and protozoa that cause chronic infections. Borrelia burgdorferi and related organisms cause Lyme disease, a multistage, systemic infection that is transmitted by Ixodes ticks and has dermatologic, neurologic, arthralgic, and constitutional manifestations in humans and other animals. Lyme disease Borrelia have an elaborate antigenic variation system consisting of vlsE, a gene expressing a surface-exposed lipoprotein, and a series of vls silent cassettes, which serve as the source of sequences for random, segmental gene conversion events that change the sequence of the central cassette region of vlsE. It has been established that vlsE expression and variation are required for persistence of infection and immune evasion, yet the mechanisms that govern this biologically important process are poorly understood. In this project, several approaches will be used to address these questions. In Specific Aim 1, a newly developed tissue explant model system will be optimized and utilized to study vlsE recombination. The effects of environmental conditions on vlsE expression and variation will be analyzed by quantitative polymerase chain reaction (qPCR) and sequence specific PCR. Specific Aim 2 involves the systematic screening and characterization of trans-acting factors (e.g. proteins) that affect vlsE recombination and expression. A transposon mutant library will be used to screen for genes important in vlsE variation and regulation, and in vitro expression libraries will be employed to screen for factors that regulate vlsE expression and variation. In Specific Aim 3, the roles of cis-acting elements (vls-associated DNA sequences) in vlsE recombination and functional activities will be examined using a combination of bioinformatic analysis of sequences, comparison of the properties of divergent vls systems, and mutagenic analysis of DNA sequences thought to be important in VlsE sequence variation and function. These studies will define the importance and mechanistic properties of VlsE in the immune evasion, persistent infection, and pathogenesis of Lyme disease Borrelia.